New blood vessel formation, or angiogenesis, is requirement for the repair and regeneration of damaged body tissues. This process requires the coordinated activity of multiple cell types including endothelial cells, smooth muscle and other mesenchymal cells, as well as cells of the monocyte/macrophage lineage. A great amount of work has been put into identifying growth factors that can stimulate angiogenesis for therapeutic purposes. Much attention has been focused on the molecule VEGF (vascular endothelial growth factor) because of its central role in the process.
VEGF is believed to be a very specific stimulator of endothelial cells that line the interior of blood vessels. Augmentation of motility, mitogenesis, and differentiation of endothelial cells can be demonstrated in response to VEGF readily in cell culture. In addition, it has been shown that animals lacking the gene for VEGF die early in development from a failure of blood vessel formation. Thus, it is believed that VEGF could be used for therapeutic purposes to stimulate angiogenesis, for example, in treatment of heart disease and peripheral vascular insufficiency. However, it has been shown that in some instances delivery of VEGF to ischemic tissue leads to the formation of very weak, leaky blood vessels lacking the support needed from the other cell types. In these instances, VEGF alone was not able to stimulate the formation of entire mature, therapeutically valuable, blood vessels. Without being bound by any theory, it is believed that a single growth factor alone is not sufficient to stimulate the coordinated activity of all of the necessary cell types to bring about angiogenesis for therapeutic purposes, e.g., in wound healing.
Wound healing is a complex cascade of cellular and biochemical events which lead to wound closure and repair of tissues. Conventionally, three successive phases are classically distinguished in the wound healing process: (1) the inflammatory phase, which corresponds to increased vascular permeability and migration of leukocytes and macrophages; (2) the proliferative phase, characterized by, among others, fibroblast proliferation and collagen synthesis resulting in granulation tissue formation; and (3) the remodeling phase where collagen and granulation tissue rearrangements result in scar resorption.
One of the first events which usually occurs in a wound is blood extravasation that results in platelet aggregation and impregnation of the wound with platelet and serum constituents. Among these constituents are polypeptide growth factors, which are known to play a major role in tissue regeneration. It has been shown that platelet a granules, which are released by aggregated platelets, are one of the richest physiological source of platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β), while serum contains high amounts of insulin-like growth factor I (IGF-I), IGF-II and their binding proteins (IGF-BPs).
PDGFs include PDGF, platelet derived angiogenesis factor (PDAF), TGF-β and platelet factor-4 (PF-4), which is believed to be a chemoattractant for neutrophils. PDGF is a mitogen and chemoattractant for fibroblasts and smooth muscle cells and is a stimulator of protein synthesis in cells of mesenchymal origin, including fibroblasts and smooth muscle cells. PDGF is also a nonmitogenic chemoattractant for endothelial cells.
TGF-β is a chemoattractant for macrophages and monocytes. Depending on the presence or absence of other growth factors, TGF-β increases the tensile strength of healing dermal wounds. TGF-β also inhibits endothelial cell mitosis, and stimulates collagen and glycosaminoglycan synthesis by fibroblasts.
Other growth factors, such as epidermal growth factor (EGF), TGF-α, and heparin binding growth factors (HBGFs) such as VEGF and fibroblast growth factor (FGF) and osteogenin, are also believed to be involved in wound healing. Epidermal growth factor, which is found in gastric secretions and saliva, and TGF-α, which is made by both normal and transformed cells, are structurally related and may recognize the same receptors that mediate cell proliferation on epithelial cells. It is believed that both factors are involved in accelerating re-epithelialization of skin wounds.
The in vivo mode of action of these growth factors involves chemoattraction at the wound site, cell proliferation, and collagen synthesis. One very-interesting feature of these products is that it has been shown that some of them, for example, PDGF and IGFs, work synergistically in stimulating wound repair.
Growth factors are, therefore, potentially useful for specifically promoting wound healing and tissue repair. The addition of exogenous growth factors to a wound has been shown to increase the rate at which the wound is closed, the number of cells in the healing area, the growth of blood vessels, the total rate of deposition of collagen, and strength of the scar.
Platelets are fragments of cells that circulate in the blood and participate in the early phases of wound healing by binding to damaged tissue and releasing a large variety of factors that begin and sustain the healing process. Numerous factors, including VEGF, are known to be present in platelets that have a role in blood vessel formation making them a good source to look for therapeutically valuable mixtures. A number of studies have been done to show that isolates of growth factors from platelets can enhance the healing of dermal wounds. In addition, surgeons in a number of areas routinely harvest and concentrate platelets from the blood of patients (PRP, platelet-rich plasma) as a source of growth factors to be placed into specific areas of the body in need of tissue regeneration.
Platelet-derived wound healing formulae (PDWHF) are known. For example, a platelet extract, which is in the form of a salve or ointment for topical application, has been used by others in an attempt to facilitate wound healing. Unfortunately, most conventional PDWHFs are obtained by centrifuging whole blood to obtain a plasma rich in platelets but free of red blood cells and treating the platelet rich plasma with thrombin to stimulate the production of a releasate, which can be combined with collagen. These PDWHFs typically have a limited concentration of growth factors with high concentration of albumin (e.g., 9.9%). Accordingly, clinical utility of these conventional PDWHFs are limited. In addition, these PDWHFs typically contain essentially non-mature growth factors due to the use of thrombin in obtaining PDWHFs. Furthermore, conventional PDWHF production is relatively expensive, thereby making the cost-benefit ratio of conventional PDWHFs questionable.
Revised sentence: Unfortunately, most conventional PDWHFs are obtained by centrifuging whole blood to obtain a plasma rich in platelets but free of red blood cells and treating the platelet rich plasma them with thrombin to stimulate the production of a releasate, which can be combined with collagen
Autologous human platelet derived wound healing formula, made of thrombin activated platelet a granules, has also been shown to induce the healing of chronic ulcers, thus making growth factor extracts an advantageous alternative to the use of recombinant growth factors.
Accordingly, there is a continuing need for better wound healing compositions and methods for obtaining such compositions.